Pulmonary Expression Is Coordinately Regulated by STAT1, STAT6, and IFN-γ

This information is current as Patricia C. Fulkerson, Nives Zimmermann, Lynn M. of September 29, 2021. Hassman, Fred D. Finkelman and Marc E. Rothenberg J Immunol 2004; 173:7565-7574; ; doi: 10.4049/jimmunol.173.12.7565 http://www.jimmunol.org/content/173/12/7565 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Pulmonary Chemokine Expression Is Coordinately Regulated by STAT1, STAT6, and IFN-␥

Patricia C. Fulkerson,* Nives Zimmermann,‡ Lynn M. Hassman,‡ Fred D. Finkelman,† and Marc E. Rothenberg1‡

The expression of distinct within the asthmatic lung suggests that specific regulatory mechanisms may mediate various stages of asthmatic disease. Global transcript expression profiling was used to define the spectrum and kinetics of chemokine involvement in an experimental murine model of asthma. Seventeen chemokines were induced in the lungs of allergen-inoculated mice, as compared with saline-treated mice. Two (CXCL13 and CCL9) of the 17 identified chemokines have not previously been associated with allergic airway disease. Seven (7 of 17; CCL2, CCL7, CCL9, CCL11, CXCL1, CXCL5, CXCL10) of the allergen- induced chemokines were induced early after allergen challenge and remained induced throughout the experimental period. Three chemokines (CXCL2, CCL3, and CCL17) were induced only during the early phase of the inflammatory response after the initial Downloaded from allergen challenge, while seven chemokines (CCL6, CCL8, CCL12, CCL22, CXCL9, CXCL12, and CXCL13) were increased only after a second allergen exposure. Unexpectedly, expression of only three chemokines, CCL11, CCL17, and CCL22, was STAT6 dependent, and many of the identified chemokines were overexpressed in STAT6-deficient mice, providing an explanation for the enhanced neutrophilic inflammation seen in these mice. Notably, IFN-␥ and STAT1 were shown to contribute to the induction of two STAT6-independent chemokines, CXCL9 and CXCL10. Taken together, these results show that only a select panel of che- mokines (those targeting Th2 cells and ) is positively regulated by STAT6; instead, many of the allergen-induced http://www.jimmunol.org/ chemokines are negatively regulated by STAT6. Collectively, we demonstrate that allergen-induced inflammation involves coor- dinate regulation by STAT1, STAT6, and IFN-␥. The Journal of Immunology, 2004, 173: 7565–7574.

ne hallmark of allergic airway disease is accumulation of kines at different stages in the evolution of allergic lung inflam- eosinophils, , lymphocytes, and mation, and their regulation in vivo are only partially understood. O in the lung (1). Within the airway mucosa, CD4ϩ Th2- Murine models of allergic airway inflammation have demon- type lymphocytes (Th2 cells) and other inflammatory leukocytes strated that overexpression of products of Th2 cells, spe- release a range of inflammatory mediators that contribute both di-

cifically IL-4 and IL-13, is sufficient for the induction of numerous by guest on September 29, 2021 rectly and indirectly to remodeling of the airway wall, mucus hy- lung chemokines and the development of pulmonary eosinophilia (13, persecretion, airway obstruction, and airway hyperreactivity (2– 14). IL-4 and IL-13 share a chain, IL-4R␣, which mediates 4). Chemokines are a large family of chemotactic that phosphorylation of JAK1 and JAK3, and, subsequently, phosphory- orchestrate the migration and activation of leukocyte populations lation of IL-4R␣. STAT6 monomers are then recruited to the phos- under baseline (homeostatic) and inflammatory conditions (5–8). phorylated docking tyrosine residues in IL-4R␣ and phosphorylated This large family of cytokines has been divided into four groups, by JAKs, resulting in STAT6 dimerization and translocation to the designated CXC, CC, C, and CX3C, depending on the spacing of nucleus. STAT6 is required for many IL-4- and IL-13-mediated re- conserved cysteine residues. The CXC chemokines mainly target sponses, including CCL11 expression (15–17). Although STAT6-de- neutrophils and lymphocytes, whereas the CC chemokines target a ficient mice have attenuation of many features of experimental asthma variety of cell types, including macrophages, eosinophils, ba- (e.g., pulmonary eosinophilia), they are either only partially protected sophils, and dendritic cells. For example, CCL11 is a highly potent or not protected at all from other aspects of the disease that are less -selective chemoattractant that induces eosinophil de- specific for allergy, such as lung neutrophilia (18, 19). Surprisingly, granulation (9–12). Although extensive studies have demonstrated the mechanism by which STAT6 deficiency promotes neutrophilia a central role for chemokines in controlling multiple aspects of the has not been established. There are several proposed general mecha- asthmatic response, the full spectrum of chemokines involved in nisms by which STAT6 regulates inflammatory cell recruitment (e.g., allergic airway inflammation, the distinct role of specific chemo- it may be required for induction of specific chemokines and adhesion molecules). However, it remains to be determined exactly how STAT6 regulates inflammatory cell recruitment in experimental Departments of *Molecular Genetics, Biochemistry, and Microbiology, and †Internal Medicine, Division of Immunology, University of Cincinnati College of Medicine, asthma. This is not just an academic question, because STAT6 and its Cincinnati, OH 45257; and ‡Division of Allergy and Immunology, Cincinnati Chil- related signaling pathway are targets for drug development for dren’s Hospital Medical Center, University of Cincinnati College of Medicine, Cin- cinnati, OH 45229 asthma. As such, it is critical to characterize allergen-induced lung Received for publication April 5, 2004. Accepted for publication August 30, 2004. inflammation in the absence of STAT6, because this could be the state of patients who someday receive IL-4, IL-13, and/or STAT6 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance antagonists. with 18 U.S.C. Section 1734 solely to indicate this fact. Recently, we have taken an empiric approach to define the broad 1 Address correspondence and reprint requests to Dr. Marc E. Rothenberg, Division spectrum of associated with induction of experimental of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, Univer- sity of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229- asthma in mice (20). Of the 291 asthma signature genes identified, 3039. E-mail address: [email protected] we found overexpression of expected Th2-associated cytokines

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 7566 ASTHMA CHEMOKINE PROFILE

(IL-4, CCL11, CCL2, and CCL8); however, several Th1- and IFN- murine U74Av2 GeneChip (Affymetrix, Santa Clara, CA), the chips ␥-associated chemokines were also up-regulated. Focusing on the were automatically washed and stained with streptavidin-PE using a flu- chemokines CXCL9 and CXCL10, we have demonstrated that idics system. The chips were scanned with a Hewlett-Packard GeneArray Scanner (Palo Alto, CA). This analysis was performed with one mouse per they negatively regulate eosinophil lung recruitment and function chip (n Ն 3 for each allergen challenge condition, and n Ն 2 for each saline (21). This finding highlights the complex interaction between nu- challenge condition). merous chemokines in the setting of allergic airway inflammation. Additionally, the presence of IFN-␥- and IL-4/IL-13-associated Northern blot analysis chemokines within the asthmatic lung suggests the interplay of RNA was electrophoresed in an agarose-formaldehyde gel, transferred to Gene intricate regulatory mechanisms, which have not yet been fully Screen transfer membranes (NEN, Boston, MA) in 10ϫ SSC, and cross-linked elucidated. In this study, we took a global approach to identify by UV radiation, as previously reported (24, 25). The cDNA probes, generated chemokines associated with the murine model of experimental by PCR or from commercially available vectors (I.M.A.G.E. Consortium ob- asthma. Furthermore, we aimed to dissect the coordinated kinetic tained from American Tissue Culture Collection (Manassas, VA) or Incyte Genomics (Palo Alto, CA)), were sequence confirmed, radiolabeled with 32P, expression and transcriptional regulation of these chemokines. We and hybridized using standard conditions. identified 17 allergen-induced chemokines and demonstrated that they are regulated by distinct kinetic patterns. Two (CXCL13 and Microarray data analysis CCL9) of the 17 identified chemokines have not previously been associated with allergic airway disease. In addition, we report a From data image files, gene transcript levels were determined using algo- rithms in the Microarray Analysis Suite software (Affymetrix). Global scal- profound negative regulatory role for STAT6 on the expression of ing was used to compare genes from chip to chip; thus, each chip was several chemokines in the allergic lung; 7 of the 17 chemokines normalized to an arbitrary value (1500). Differences between saline- and Downloaded from were surprisingly enhanced following allergen challenge in the allergen-treated mice were also determined using GeneSpring software absence of STAT6. Only expression of 3 chemokines, CCL11, (Silicon Genetics, Redwood City, CA). Data were normalized to the av- CCL17, and CCL22, was STAT6 dependent. Also, expression of 2 erage of the saline-treated mice. Gene lists were created, as previously reported (20). Analysis of chemokine induction was performed from pre- chemokines, CXCL9 and CXCL10, was dependent on the Th1- viously reported gene lists (20). Fold increase for each chemokine was associated cytokine and IFN-␥ and STAT1, calculated by dividing the mean of the average difference values of the ϭ respectively. Taken together, these results demonstrate that aller- OVA-challenged mice (n 3) by the mean of the average difference values http://www.jimmunol.org/ ϭ gic airway inflammation is accompanied by the interplay of a large of the saline-challenged mice (n 2) at each of the time points. Relative fold change over time was calculated by dividing the mean of the average number of chemokines with distinct kinetic and transcriptional difference values at 18 h after the first and second OVA challenge by the regulation. Notably, despite the central role of IL-4, IL-13, and mean of the average difference values at 3 h after the first OVA challenge STAT6 in promoting those aspects of airway inflammation that are for each of the chemokines. specific for allergy and asthma, these molecules are not responsible for global induction of the majority of chemokines in the allergen- BALF collection and analysis challenged lung; rather they, directly or indirectly, reduce expres- Mice were euthanized by CO2 inhalation. A midline neck incision was sion of several chemokines that promote a less specific inflamma- made, and the trachea was cannulated. The lungs were lavaged twice with tory response. 1.0 ml of PBS containing 1% FCS and 0.5 mM EDTA. The recovered by guest on September 29, 2021 BALF was centrifuged at 400 ϫ g for 5 min at 4°C, and resuspended in 200 ␮l. Lysis of RBC was conducted using RBC lysis buffer (Sigma-Aldrich, Materials and Methods St. Louis, MO), according to the manufacturer’s recommendations. Total Experimental asthma induction cell numbers were counted with a hemacytometer. Cytospin preparations of 1 ϫ 105 cells were stained with the Hema 3 Staining System (Fisher Di- BALB/c mice were obtained from the National Cancer Institute (Frederick, agnostics, Middletown, VA), and differential cell counts were determined. MD), STAT6-deficient mice (BALB/c background) from The Jackson Lab- oratory (Bar Harbor, ME), and STAT1-deficient mice (SVEV background) from Taconic Farms (Germantown, NY). All mice were housed under spe- Cytokine quantitation cific pathogen-free conditions. Asthma-like lung disease was induced by two i.p. injections with 100 ␮g of OVA adsorbed to 1 mg of aluminum Cytokine concentration in the BALF of allergen- and saline-chal- hydroxide (alum), followed by two 50 ␮g of OVA or saline intranasal lenged mice was quantified by using ELISA kits specific for CXCL9 (R&D Systems, Minneapolis, MN); the detection limit was 0.9 pg/ml. IFN-␥ con- challenges 3 days apart, as previously described (22). Mice were sacrificed ␥ ␮ 3 or 18 h following the first or second allergen challenge. Wild-type and centration was measured using rat mAbs to mouse IFN- (5 g/ml clone R4-6A2, 1 ␮g/ml biotinylated clone XMG1.2; BD Pharmingen, San Diego, STAT6-deficient mice were treated with 1 mg of a neutralizing monoclonal ␥ anti-murine IFN-␥ (XMG-6) (23) or rat IgG1 control mAb 24 h before the CA); detection limit was 8 pg/ml for IFN- . first allergen challenge. Subsequently, the bronchoalveolar lavage fluid (BALF)2 and/or lung tissue were harvested 18–24 h after challenge. The Results left lobe of the lungs was fixed in Formalin, embedded in paraffin, and stained with H&E using standard histological techniques. Experimental asthma induction We were first interested in identifying genes that were differen- Preparation of RNA and microarray hybridization tially expressed in a well-established model of experimental RNA was extracted using the TRIzol reagent as per the manufacturer’s asthma. In our model, mice were i.p. sensitized with the allergen instructions. Following TRIzol purification, RNA was repurified with phe- OVA in the presence of the adjuvant alum on two occasions sep- nol-chloroform extraction and ethanol precipitation. Microarray hybridiza- tion was performed by the Affymetrix Gene Chip Core facility at Cincin- arated by 14 days (Fig. 1A). Subsequently, mice were challenged nati Children’s Hospital Medical Center. Briefly, RNA quality was first with intranasal OVA or control saline on two occasions separated assessed using the Agilent bioanalyzer (Agilent, Palo Alto, CA), and only by 3 days. Three or 18 h after the first allergen challenge and 18 h those samples with 28S/18S ratios between 1.3 and 2 were subsequently after the second allergen challenge, the lungs were examined for used. RNA was converted to cDNA with Superscript choice for cDNA synthesis (Invitrogen Life Technologies, Carlsbad, CA), and subsequently inflammatory cellular infiltration or harvested for RNA analysis. converted to biotinylated cRNA with Enzo High Yield RNA Transcript Total cell numbers recovered from the airway lumen were signif- labeling (Enzo Biochem, Farmingdale NY). After hybridization to the icantly increased (from 5.2 Ϯ 0.9 ϫ 104 to 29.9 Ϯ 12.7 ϫ 104) following allergen challenge compared with saline controls, and 2 Abbreviations used in this paper: BALF, bronchoalveolar lavage fluid; SOCS, sup- the presence of neutrophils, lymphocytes, and, most dramatically, pressor of cytokine signaling. eosinophils was increased in the airway of the allergic lung (Fig. The Journal of Immunology 7567

FIGURE 1. Murine model of experimental asthma. A, A schematic representation of the al- lergen challenge protocol is depicted. Mice re- ceived two i.p. injections of OVA and alum. Subsequently, mice were challenged with OVA or saline intranasally and analyzed 3 or 18 h after the first or second allergen challenge. B, Cellular composition of BALF 18 h after the second OVA challenge. A representative experiment p Ͻ ,ء .n ϭ 2) with four mice/group is shown) 0.01. C, H&E-stained saline- and allergen-chal- lenged lungs (18 h after second OVA challenge); magnification ϭϫ100. Higher power magnifi- cation (ϫ400) shows infiltration around lung blood vessel. Downloaded from

1B). Examination of the lung demonstrated profound peribronchial genes, it was notable that chemokines represented a large subset; http://www.jimmunol.org/ and perivascular inflammation (Fig. 1C). 17 of the 28 chemokines represented on the chip were induced compared with saline-challenged control mice. Seven CXC and 10 Identification of the chemokine panel expressed in experimental CC chemokines were induced 2-fold or more at their peak of ex- asthma pression compared with saline-challenged mice (Fig. 2A). Leukocyte recruitment to the lung during allergic inflammation is We next analyzed the chromosomal location of the chemokines partially dependent on chemokines (8, 26). We were interested in represented on the chip and the chemokines induced following identifying a broad spectrum of chemokines that contributed to the allergen challenge. Transcripts from 5, 6, 8, and 11

marked inflammatory response in our experimental allergic airway were increased with allergen challenge. Most of the members of by guest on September 29, 2021 model. Lung RNA was subjected to microarray analysis using the the CXC chemokine loci on 5 (CXCL1, CXCL2, Affymetrix chip U74Av2 that contains oligonucleotide probe sets CXCL5, CXCL9, CXCL10, and CXCL13) that were present on representing 12,422 genetic elements (20). Of the allergen-induced the chip were among the increased chemokine genes. In addition,

FIGURE 2. Identification of allergen-induced chemokines. A, Peak fold increase for allergen- induced CC and CXC chemokines with a mean average difference fold increase of Ͼ2 in aller- gen-challenged lung in comparison with saline- challenged control lungs (n ϭ 2 for saline-chal- lenged lungs; n ϭ 3 for OVA-challenged lungs). B, Chemokine expression at three different time points in asthma model. Gene lists of chemo- kines induced Ͼ2-fold in the allergen-chal- lenged lung in comparison with saline-chal- lenged lung were generated at 3 h after the first OVA challenge, and 18 h after the first and sec- ond OVA challenge. Overlap of the gene lists is represented in the Venn diagram format. 7568 ASTHMA CHEMOKINE PROFILE

8 of the 11 CC chemokines included in the CC chemokine cluster CCL12, CCL22, CXCL9, CXCL12, and CXCL13; Fig. 2B). This on were among the increased genes. analysis revealed that chemokines are coregulated (kinetically) de- spite diverse structure and chromosomal locations. Stratification of allergen-induced chemokines by their kinetic To gain a better understanding of the temporal regulation of induction profile each chemokine, we examined the change in chemokine expres- We were next interested in testing the hypothesis that related che- sion from an early point after the first allergen challenge through mokines would be induced with similar kinetic expression pat- a later time point following the second allergen challenge. Con- terns. To test this, we examined chemokine expression at three sistent with changes in BALF cellular infiltration over time, three different time points in our asthma model. Several (7 of 17; CCL2, distinct temporal patterns of chemokine expression were identi- CCL7, CCL9, CCL11, CXCL1, CXCL5, CXCL10) of the aller- fied: early (Fig. 3A), late (Fig. 3C), and stable (Fig. 3E, and data gen-induced chemokines were induced 2-fold or more early after not shown). Five chemokines (CXCL1, CXCL2, CXCL5, CCL3, the first allergen challenge when compared with saline controls and CCL17) were grouped in the early induction pattern with peak and remained induced throughout the experimental period (Fig. expression occurring 3 h after the first allergen challenge (Fig. 3A). 2B). Three chemokines (CXCL2, CCL3, and CCL17) were in- Four of these chemokines, CXCL1, CXCL2, CXCL5, and CCL3, duced only early on in the allergic inflammatory response, while are potent chemoattractants in the mouse, consistent another large subset of the allergen-induced chemokines was in- with the early influx of neutrophils into the airway following the creased only after the second allergen challenge (CCL6, CCL8, initial allergen challenge (data not shown). Northern blot analysis Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Temporal regulation of allergen-induced chemokines. Analysis of the mean average difference fold change of each chemokine at later time points (18 h after first and second OVA challenge) in asthmatic lung in relation to the early time point (3 h after first OVA challenge) revealed three distinct temporal profiles of chemokine expression: early induction (A) chemokines, the late induction (C) chemokines, and the stable induction (E) chemokines. Northern blot analysis (B, D, and F) confirmed expression of representative chemokines from each profile. The Journal of Immunology 7569 confirmed the early induction of CXCL1, CXCL2, and CXCL5 of STAT6 (Fig. 4B). These results offer a possible explanation for (Fig. 3B). The late pattern of chemokine induction in the allergic the inflammation seen in allergen-inoculated STAT6-deficient pulmonary response was characterized by the induction of seven mice. Indeed, while STAT6-deficient mice had reduced lung eo- chemokines (CCL6, CCL8, CCL9, CCL11, CCL12, CXCL9, and sinophilia, there was an increase in BALF neutrophils following CXCL13) with their expression increasing over time and their allergen challenge (Fig. 4C). Examination of the lung tissue re- peak expression occurring 18 h after the second allergen challenge vealed that modest levels of allergen-induced peribronchial and (Fig. 3C). Peak expression of eosinophil active chemokines (CCL8 perivascular inflammation were present in STAT6-deficient mice and CCL11) in the late induction pattern is consistent with the (Fig. 4D). Taken together, these results demonstrate that STAT6 is eosinophil-rich inflammatory cell accumulation 18 h after the sec- required for induction of Th2-specific chemokines, CCL17, ond allergen challenge (Fig. 1B). Northern blot analysis confirmed CCL22, and CCL11. In addition, STAT6 acts as a negative regu- the increasing expression of CCL6 and CCL11 over time in the lator (directly or indirectly) of expression for chemokines associ- allergic lung (Fig. 3D). The expression of five chemokines (CCL2, ated with a general inflammatory response. As such, certain as- CCL7, CCL22, CXCL10, and CXCL12) did not change Ͼ2-fold in pects of the inflammatory process might be exaggerated in the relation to their expression at the early time point in our asthma absence of STAT6. model (Fig. 3E). Northern blot analysis confirmed the relatively steady induction of CCL2 and CCL22. We have previously re- ␥ ported a very high correlation between our gene chip data and IFN- is required for the expression of the allergen-induced Northern blot analysis of RNA levels (27). Indeed, 10 of the 12 chemokines CXCL9 and CXCL10 chemokines probed by Northern blot analysis were completely In the classic model of allergen-induced chemokine expression in Downloaded from consistent with the gene chip data (Fig. 3, and data not shown). the lung, the Th2 cytokines IL-4 and IL-13 have a dominant role CCL2, by Northern blot analysis, was modestly different, revealing in orchestrating inflammatory cell recruitment. Our finding that an early pattern of induction rather than steady induction derived Th1-associated chemokines were also increased during the induc- from the gene chip analysis. However, it is important to note that tion of experimental asthma and that STAT6 had a prominent in- CCL2 was indeed still induced after the early time point (Fig. 3F). hibitory role implicated a role for IFN-␥ in our experimental

Additionally, CXCL12 induction was more comparable with a late model. Indeed, several studies have proposed that the asthmatic http://www.jimmunol.org/ induction pattern (data not shown). Although both Northern blot lung is associated with Th1- and Th2-regulated processes (29). and gene chip analysis are based on two different techniques to Consistent with this, many chemokines from our panel of allergen- assess mRNA levels, it is remarkable how well they correlated. induced chemokines have been shown to be induced by IFN-␥ Taken together, these data indicate that a large panel of chemo- (e.g., CXCL1, CXCL2, CXCL9, CXCL10, CCL2, CCL3, CCL8). kines is induced in the allergic lung, and that expression of the We therefore measured IFN-␥ protein levels in the BALF of OVA- chemokines is temporally controlled. challenged wild-type and STAT6-deficient mice. Although IFN-␥ levels in the airways of wild-type mice remained below the level STAT6 positively and negatively regulates allergen-induced of detection (8 pg/ml), IFN-␥ was significantly increased from chemokines undetectable to 51.5 Ϯ 17.5 pg/ml (mean Ϯ SD, n ϭ 4 mice/ by guest on September 29, 2021 Having identified a profile of chemokines associated with allergic group) in the BALF of OVA-challenged STAT6-deficient mice airway responses, we were next interested in identifying factors compared with saline-challenged mice. To test the hypothesis that involved in regulating their expression. We hypothesized that IFN-␥ was contributing to chemokine induction in the asthmatic STAT6 would be required for the induction of many chemokines lung, we treated OVA-sensitized mice with a neutralizing anti- in the asthmatic lung because this is an essential transcription fac- IFN-␥ mAb before the first allergen challenge, and then examined tor for the development of Th2-associated responses (15, 18, 28). the levels of leukocytes and chemokine expression in the late- However, we surprisingly found that most (14 of 17) allergen- phase lung (18 h after the second OVA challenge). The ability of induced chemokines did not require STAT6 for their expression; in the Ab to neutralize IFN-␥ was confirmed by the reduction of fact, 7 of the 14 STAT6-independent chemokines were expressed IFN-␥ protein levels in the BALF 18 h after the second OVA more in the absence of STAT6. For example, the mRNA level of challenge (IFN-␥ levels in the BALF of OVA-challenged STAT6- an early CXC chemokine (CXCL2) was enhanced in the absence deficient mice were Ͻ8 pg/ml after anti-IFN-␥ treatment). Next, of STAT6 following allergen challenge (Fig. 4A). Furthermore, we examined the effect of anti-IFN-␥ treatment on the levels of expression of all of the allergen-induced late and stable pattern allergen-induced chemokines in the lung. Allergen-induced ex- CXC chemokines was increased, when compared with wild-type pression of the chemokines CXCL9 and CXCL10 was profoundly control mice, suggesting that STAT6 acts as a negative regulator of reduced in both wild-type and STAT6-deficient mice following this family of chemokines, especially later in the developing al- anti-IFN-␥ treatment compared with mice treated with an isotype lergic inflammatory response (Fig. 4A). Consistent with the en- control Ab (Fig. 5A). Consistent with the reduction in mRNA ac- hanced transcription in the absence of STAT6, analysis of protein cumulation, protein levels of CXCL9 were dramatically reduced in expression revealed an increase in CXCL9 in the lungs of OVA- the BALF of OVA-challenged mice treated with anti-IFN-␥ com- challenged STAT6-deficient mice (1882 Ϯ 421 pg/ml; mean Ϯ pared with control IgG treatment in wild-type (35.9 Ϯ 19.7 vs SD, n ϭ 4 mice/group) in comparison with wild-type (280 Ϯ 154 128.4 Ϯ 43.9 pg/ml) and in STAT6-deficient mice (113.8 Ϯ 30.5 pg/ml; mean Ϯ SD, n ϭ 4 mice/group). We also examined the vs 1142.5 Ϯ 822.2 pg/ml). Expression of the CXC chemokines expression of allergen-induced CC chemokines in STAT6-defi- CXCL12 and CXCL13 was unaffected by inhibition of IFN-␥ in cient mice. Like most of the CXC chemokines, expression of the allergic lung (Fig. 5A), indicating that the overexpression of CCL3 and CCL12 was enhanced in the absence of STAT6 (Fig. these two chemokines in STAT6-deficient mice was not IFN-␥ 4B). Although transcription of allergen-induced CCL2, CCL6, dependent. We also examined the role of IFN-␥ in allergen-in- CCL7, CCL8, and CCL9 was similar in wild-type and STAT6- duced expression of several CC chemokines, including CCL2, deficient lungs, only eosinophil-specific chemokine, CCL11, ex- CCL8, CCL9, CCL11, and CCL12. Expression of the eosinophil- pression and Th2 chemoattractants, CCL22 and CCL17, specific chemokine CCL11 and the other CC chemokines was un- expression were significantly reduced or eliminated in the absence changed by anti-IFN-␥ treatment (Fig. 5B). 7570 ASTHMA CHEMOKINE PROFILE Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. STAT6 regulation of allergen-induced chemokines. A, STAT6 negatively regulates most CXC chemokines. B, STAT6 is required for only 3 of 10 allergen-induced CC chemokines. Northern blot analysis of CC and CXC chemokine expression in STAT6-deficient lungs is shown. Ethidium bromide (EtBr)-stained gel is also shown. Each lane represents a separate mouse. C, Cellular composition of the BALF in the STAT6-deficient lung is ,p Ͻ 0.02. D ,ء .shown following induction of experimental asthma. A representative experiment is shown (n ϭ 2) with three to four mice in each group H&E-stained STAT6-deficient allergic lungs (18 h after second OVA challenge); magnification ϭϫ100.

Finally, we were interested in determining whether the ability of mice because IFN-␥ signaling is partly dependent on this tran- anti-IFN-␥ to inhibit CXCL9 and CXCL10 was associated with a scription factor (30). Notably, IFN-␥-induced expression of some change in the infiltration of leukocytes into the allergic airway. chemokines has been shown to be STAT1 independent in vitro, Based on our prior findings that these chemokines negatively reg- highlighting the importance of analyzing the role of STAT1 in ulate eosinophil recruitment into the lung, we hypothesized that vivo (30). To determine whether STAT1 has a prominent role in anti-IFN-␥ would increase the level of BALF eosinophils. Indeed, the regulation of IFN-␥-dependent chemokine expression in the anti-IFN-␥ treatment increased eosinophil levels in both wild-type allergic lung, we induced allergic airway disease in STAT1-defi- and STAT6-deficient mice in comparison with control IgG-treated cient mice. BALF eosinophils were significantly increased not mice (Fig. 5C). An increase in allergen-induced airway neutro- only with allergen challenge in the STAT1-deficient mice, but also philia was also observed in the STAT6-deficient mice following in comparison with OVA-challenged wild-type control mice (Fig. anti-IFN-␥ treatment, while mononuclear cells, monocytes, and 6A). We next examined IFN-␥-dependent chemokine expression in lymphocytes remained unchanged (Fig. 5C, and data not shown). the lungs of STAT1-deficient mice following allergen challenge. These findings demonstrate that IFN-␥ functions as a regulator of Allergen-induced expression of CXCL9 and CXCL10 was com- infiltrating leukocytes into the allergic lung. In particular, IFN-␥ pletely absent in the lungs of STAT1-deficient mice, demonstrating negatively regulates eosinophil recruitment into the lung by induc- the importance of this transcription factor in the expression of the ing expression of the inhibitory chemokines CXCL9 and CXCL10. eosinophil inhibitory chemokines CXCL9 and CXCL10 in the asth- matic lung (Fig. 6B). Also, protein levels of CXCL9 in the BALF of STAT1 regulation of allergen-induced chemokines OVA-challenged STAT1-deficient mice were below the level of de- To further characterize the role of IFN-␥ in allergen-induced che- tection, confirming the importance of this transcription factor in the mokine expression in the lung, we also examined STAT1-deficient induction of CXCL9 in response to allergen challenge. Together, The Journal of Immunology 7571

FIGURE 5. IFN-␥ regulates expression of CXCL9 and CXCL10. A and B, Northern blot analysis of CXC (A) and CC (B) che- mokine expression in allergic lung after treatment with anti-IFN-␥ or control IgG1 (cIgG) is shown. Ethidium bromide-stained gel is also shown. Each lane represents a sep- Downloaded from arate mouse. C, Eosinophils and neutrophils in the BALF from wild-type (WT; Ⅺ) and STAT6-deficient (f) mice after induction of experimental asthma. A representative ex- periment is shown (n ϭ 2) with three to four .p Ͻ 0.04 ,ء .mice in each group http://www.jimmunol.org/ by guest on September 29, 2021

these data suggest a role for IFN-␥ and STAT1 in the regulation of sented on the chip used in our analysis. Second, we demonstrate eosinophil accumulation in allergic inflammation because they are that STAT6 up-regulates only a select group of chemokines asso- important for expression of the eosinophil inhibitory chemokines ciated with eosinophilia and Th2 cell recruitment, while most of CXCL9 and CXCL10. the chemokines expressed in the allergic lung are STAT6 inde- pendent. Third, we demonstrate that the infiltration of leukocytes, Discussion in particular eosinophils, is regulated by the coordinated interplay Chemokines, produced by both airway epithelial and infiltrating of STAT6, IFN-␥, and STAT1. And fourth, our results offer an cells, are believed to orchestrate the accumulation and activation of explanation for the allergen-induced inflammation that develops in different leukocyte populations in the lung (12, 31). In this study, STAT6-deficient mice because we have determined that a large we aimed to identify the spectrum of chemokines expressed in the number of allergen-induced chemokines are negatively regulated murine lung in response to allergen challenge. To further under- by STAT6. This later finding identifies a pathway that explains the stand the role of each chemokine in the evolution of the allergic allergen-induced inflammation seen in the absence of classic Th2 response, we focused on the kinetic expression profile for each . allergen-induced chemokine. In addition, we examined the contri- To our knowledge, this is the first report of an overwhelmingly bution of the cytokine IFN-␥ and the transcription factors STAT1 negative regulatory role for STAT6 in chemokine expression in the and STAT6 in regulating the expression of these chemokines (Ta- ble I). Although considerable information is known about chemo- allergic lung. The downstream effects of STAT6 deficiency in the kines in allergic lung disease, in this study, we have uncovered developing allergic response are complex. It is important to note several new findings with biological significance. First, in addition that the absence or presence of STAT6 had both dramatic effects to identifying a panel of 17 chemokines temporally induced during on chemokine expression (e.g., CCL11 and CXCL13) as well as the development of experimental asthma, we have identified two subtle consequences (e.g., CCL17 and CCL22). As such, we can- chemokines that have not been previously associated with allergic not rule out the possibility that the inhibitory effects of STAT6 are airway disease. Although our global analysis of chemokine induc- indirect because chemokine mRNA levels can be modified by a tion in the allergic lung is extensive, it is not exhaustive because variety of mechanisms, including mRNA stability (32). In addi- several murine chemokines (CXCL3, CXCL6, CXCL11, tion, Th2 cytokines, IL-4 and IL-13, have been shown to induce CXCL15, CCL16, CCL20, CCL24, and CCL28) were not repre- the expression of suppressors of cytokine signaling (SOCS)1 in a 7572 ASTHMA CHEMOKINE PROFILE

Table I. Allergen-induced chemokine regulation by STAT6, IFN-␥, and STAT1a

Allergen-Induced Chemokinesb STAT6 IFN-␥ STAT1

Early induction pattern CCL3 MIP-1␣ Ϫ ND ND CCL17 TARC ϩ ND ND CXCL1 GRO␣ NE ND ND CXCL2 GRO␤ Ϫ ND ND CXCL5 LIX NE ND ND

Late induction pattern CCL6 C10 NE NE ND CCL8 MCP-2 NE NE NE CCL9 MRP-2 NE NE NE CCL11 Eotaxin-1 ϩ NE ND CCL12 MCP-5 Ϫ NE ND CXCL9 MIG Ϫϩϩ CXCL13 BLC Ϫ NE ND

Stable induction pattern Downloaded from CCL2 JE NE NE ND CCL7 MCP-3 NE NE ND CCL22 ABCD-1 ϩ NE ND CXCL10 IP-10 Ϫϩϩ CXCL12 SDF-1 Ϫ NE ND

a If expression of the chemokine is reduced or eliminated in the absence or re- FIGURE 6. STAT1 is required for allergen-induced expression of duction of the cytokine or transcription factor, the chemokine is labeled with “ϩ” for http://www.jimmunol.org/ CXCL9 and CXCL10. A, BALF cellular composition in wild-type (WT) positive regulation. If the chemokine mRNA levels are increased in the absence of Ϫ and STAT1-deficient lungs after induction of experimental asthma. A rep- STAT6, the chemokine is labeled “ ” for negative regulation. If mRNA levels are ϭ unaffected with the depletion or loss of the transcription factor or cytokine, the che- resentative experiment is shown (n 3) with three to four mice in each mokine is labeled with “NE” for no effect. -p Ͻ 0.03. B, Northern blot analysis of CXCL9 and CXCL10 b TARC, and activation-regulated chemokine; GRO, growth-related on ,ء .group expression in wild-type (WT) and STAT1-deficient lungs following saline cogene; LIX, LPS-inducible CXC chemokine; MRP, MIP-related protein; MIG, ␥ ␥ and allergen (OVA) challenge is shown. Ethidium bromide-stained gel is induced by IFN- ; BLC, B lymphocyte chemoattractant; IP-10, IFN- - inducible protein of 10 kDa; SDF, stromal cell-derived factor. also shown. Each lane represents a separate mouse. by guest on September 29, 2021 lung cell line, and SOCS1 has been demonstrated to regulate cy- mediated regulation of its receptor, CXCR4, has been examined in tokine-induced chemokine expression by fibroblasts (33–36). In eosinophils and lung epithelial cells, regulation of CXCL12 in in- our experimental asthma model, SOCS1 and SOCS3 expression flammatory processes remains unclear (44, 45). The CXCL12/ was induced 18 h after the first and second allergen challenge in CXCR4 axis has been shown to contribute to the inflammatory wild-type mice, but there was no significant induction in the response in a murine model of asthma (46). The level of CXCL12 STAT6-deficient asthmatic lungs (27). Thus, we can speculate that protein, as measured by immunohistochemistry, was reported as the STAT6-dependent inhibition of chemokine expression in al- unchanged, but mRNA levels were unexamined (46). Another con- lergic airway disease may be mediated, at least in part, by SOCS sequence of STAT6 deficiency in the allergic lung may include . Our data are surprising because prior work has shown altering the effector activity of infiltrating leukocytes due to the that IL-4 and IL-13 can induce a variety of chemokines (in addi- increased chemokine levels. Indeed, we have found the induction tion to CCL11, CCL17, and CCL22) (37). Thus, while IL-4 and of numerous other inflammation-associated genes in the allergen- IL-13 overexpression is sufficient for induction of CCL2, CCL6, challenged lungs of STAT6-deficient mice (27) (data not shown). CCL7, CCL8, and CCL12, it is not required for the induction of Notably, these genes include -associated transcripts, these chemokines in experimental allergic airway inflammation (at such as platelet-activating factor acetylhydrolase, complement least in the OVA model) (37). component C1q, and CCR2. In addition, 19 allergen-induced genes Although the negative effects of STAT6 on IFN-␥ production were unique to the STAT6-deficient asthmatic lungs when com- and signaling are well-established, as are the positive effects of the pared with wild-type controls, including several lymphocyte-asso- IFNs and STAT1 on CXCL9 and CXCL10 in other models, our ciated transcripts (27). Additional studies are needed to determine data demonstrating overexpression of several chemokines in the relationship between chemokine expression and the new ge- STAT6-deficient mice suggest that these pathways actually con- netic program induced in STAT6-deficient mice. tribute to a specific type of inflammation in allergen-treated mice In our experimental model, IFN-␥ induces the expression of that only occurs in the absence of STAT6 (30, 38–40). For ex- CXCL9 and CXCL10, by a STAT1-dependent mechanism. These ample, enhanced neutrophil-active chemoattractant expression results implicate an important role for the Th1-associated cytokine (e.g., CXCL2 and CCL3) may lead to increased cell activation and IFN-␥ and signaling molecule STAT1 in the regulation of chemo- mediator release, perpetuating the inflammatory response and con- kine expression and ultimately the recruitment of inflammatory tributing to tissue damage in lung diseases, such has been shown cells, especially eosinophils, in the developing allergic response in in chronic obstructive pulmonary disease (41, 42). CXCL12, an- the lung. These collective results suggest the codevelopment of other chemokine with enhanced expression in the absence of both Th1 and Th2 responses during allergic airway inflammation. STAT6, has been demonstrated to be constitutively expressed in a Indeed, although asthma is a Th2-associated disease, numerous wide range of tissues, including the lung (43). 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